Global cerebral ischemia due to cardiac arrest results in debilitating neurological impairment necessitating costly long-term health care. Despite this major clinical and economic impact, there is currently no specific medical therapy. Global cerebral ischemia is associated with extensive necrotic and apoptotic cell death; these processes are tightly regulated by several mechanisms, including a critical role for protein phosphorylation. While the involvement of protein kinases in the control of cell death in global cerebral ischemia is well established, the role of protein phosphatases has received relatively little attention. Protein phosphatase-1 is a member of the serine/threonine protein phosphatase subfamily that has been implicated in the regulation of cell death. We have identified and purified several novel multimeric forms of protein phosphatase-1 in mammalian brain. The activity of one of these, termed protein phosphatase-1IC (PP-1IC), is activated in vivo in pig and dog models of global cerebral ischemia as well as in cell culture models of ischemia. Based on these novel findings, we hypothesize that PP-1IC is a component of the signal transduction pathways that link global cerebral ischemia to cell death. In order to determine the mechanisms of activation and the role of PP-1IC in global cerebral ischemia, we propose to purify and characterize the PP-1IC holoenzyme from control and ischemic pig forebrain following cardiac arrest with resuscitation and reperfusion. The molecular compositions of native PP-1IC purified from control and ischemic brain will be determined by mass spectrometry, and the mechanisms of PP-1IC regulation will be studied by reconstitution of the identified components in vitro. Specific membrane permeable inhibitors of PP-1 and other reagents will be developed and used to determine the role of PP-1IC as a mediator of cell death in cell culture models of cerebral ischemia. The mechanisms of ischemic activation of PP-1IC will be investigated based on the hypothesis that the Ca2+regulated protein kinases, Ca2????dependent protein kinase II (CaMKII) and/or protein kinase C4 (PKC4) function as upstream regulators. These studies will elucidate physiological and pathophysiological mechanisms that regulate native PP-1 holoenzyme activity in brain and define the role of PP-1 in the control of cell death in global cerebral ischemia. This functional proteomics approach is targeted to the development of rational mechanism-based therapies to attenuate ischemic brain cell death, with a long-term goal of clinical translation. PUBLIC HEALTH RELEVANCE: Global cerebral ischemia due to cardiac arrest results in debilitating neurological impairment necessitating costly long-term health care. Despite an immense social, medical and economic impact, there is currently no specific pharmacological therapy. The proposed studies will elucidate physiological and pathophysiological mechanisms that regulate native protein phosphatase-1 holoenzymes in brain and determine their role in the control of cell death in global cerebral ischemia. This approach is targeted to the development of rational mechanism-based protein phosphatase-1 inhibitors to attenuate ischemic cell death and neurological injury.